Design,synthesis, and biological evaluation of new inhibitors of the endocannabinoid uptake: comparison with effects on fatty acid amidohydrolase

A new series of arachidonic acid derivatives were synthesized and evaluated as inhibitors of the endocannabinoid uptake. Most of them are able to inhibit anandamide uptake with IC(50) values in the low micromolar range (IC(50) = 0.8-24 microM). In general, the compounds had only weak effects upon CB(1), CB(2), and VR(1) receptors (K(i) > 1000-10000 nM). In addition, there was no obvious relationship between the abilities of the compounds to affect anandamide uptake and to inhibit anandamide metabolism by fatty acid amidohydrolase (FAAH; IC(50) = 30-113 microM). This indicates that the compounds do not exert their effects secondarily to FAAH inhibition. It is hoped that these compounds, particularly the most potent in this series (compound 5, UCM707, with IC(50) values for anandamide uptake and FAAH of 0.8 and 30 microM, respectively), will provide useful tools for the elucidation of the role of the anandamide transporter system in vivo.     

Design, synthesis, and in vitro evaluation of carbamate derivatives of 2-benzoxazolyl- and 2-benzothiazolyl-(3-hydroxyphenyl)-methanones as novel fatty acid amide hydrolase inhibitors

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also hydrolyzes another endocannabinoid, 2-arachidonoylglycerol (2-AG). However, 2-AG has been assumed to be hydrolyzed mainly by monoacylglycerol lipase (MAGL) or a MAGL-like enzyme. Inhibition of FAAH or MAGL activity might lead to beneficial effects in many physiological disorders such as pain, inflammation, and anxiety due to increased endocannabinoid-induced activation of cannabinoid receptors CB1 and CB2. In the present study, a total of 34 novel compounds were designed, synthesized, characterized, and tested against FAAH and MAGL-like enzyme activity. Altogether, 16 compounds were found to inhibit FAAH with half-maximal inhibition concentrations (IC50) between 28 and 380 nM. All the active compounds belong to the structural family of carbamates. Compounds 14 and 18 were found to be the most potent FAAH inhibitors, which may serve as lead structures for novel FAAH inhibitors.     

Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides

Organophosphorus (OP) compound-induced inhibition of acetylcholinesterase (AChE) and neuropathy target esterase explains the rapid onset and delayed neurotoxic effects, respectively, for OP insecticides and related compounds but apparently not a third or intermediate syndrome with delayed onset and reduced limb mobility. This investigation tests the hypothesis that fatty acid amide hydrolase (FAAH), a modulator of endogenous signaling compounds affecting sleep (oleamide) and analgesia (anandamide), is a sensitive target for OP pesticides with possible secondary neurotoxicity. Chlorpyrifos oxon inhibits 50% of the FAAH activity (IC50 at 15 min, 25 degrees C, pH 9.0) in vitro at 40--56 nM for mouse brain and liver, whereas methyl arachidonyl phosphonofluoridate, ethyl octylphosphonofluoridate (EOPF), oleyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (oleyl-BDPO), and dodecyl-BDPO give IC50s of 0.08--1.1 nM. These BDPOs and EOPF inhibit mouse brain FAAH in vitro with > or =200-fold higher potency than for AChE. Five OP pesticides inhibit 50% of the brain FAAH activity (ED50) at <30 mg/kg 4 h after ip administration to mice; while inhibition by chlorpyrifos, diazinon, and methamidophos occurs near acutely toxic levels, profenofos and tribufos are effective at asymptomatic doses. Two BDPOs (dodecyl and phenyl) and EOPF are potent inhibitors of FAAH in vivo (ED50 0.5--6 mg/kg). FAAH inhibition of > or =76% in brain depresses movement of mice administered anandamide at 30 mg/kg ip, often leading to limb recumbency. Thus, OP pesticides and related inhibitors of FAAH potentiate the cannabinoid activity of anandamide in mice. More generally, OP compound-induced FAAH inhibition and the associated anandamide accumulation may lead to reduced limb mobility as a secondary neurotoxic effect.     

Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate

There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH.In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for anandamide. It is concluded that VDM11 is an inhibitor of FAAH under the assay conditions used here, and that the inhibition may at least in part be a consequence of the compound acting as an alternative substrate.     

Tetrahydrolipstatin analogues as modulators of endocannabinoid 2-arachidonoylglycerol metabolism

A series of 21 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the formation or hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Three of the novel compounds, i.e., 11, 13, and 15, inhibited 2-AG formation via the diacylglycerol lipase alpha (DAGLalpha) with IC 50 values lower than 50 nM (IC 50 of THL = 1 microM) and were between 23- and 375-fold selective vs 2-AG hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB 1 and CB 2 receptors and anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16, and 18, were slightly more potent than THL against DAGLalpha and appreciably selective vs MAGL, CB receptors, and FAAH (15-26-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity (IC 50 = 0.41 microM), and relatively ( approximately 7-fold) selective vs the other targets tested.     

Design,microwave‐assisted synthesis,biological evaluation and molecular modeling studies of 4‐phenylthiazoles as potent fatty acid amide hydrolase inhibitors

Endocannabinoids, anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), are endogenous lipids that activate cannabinoid receptors. Activation of these receptors produces anti‐inflammatory and analgesic effects. Fatty acid amide hydrolase (FAAH) is a membrane enzyme that hydrolases endocannabinoids; thus, inhibition of FAAH represents an attractive approach to develop new therapeutics for treating inflammation and pain. Previously, potent rat FAAH inhibitors containing 2‐naphthyl‐ and 4‐phenylthiazole scaffolds were identified, but up to the present time, very little structure–activity relationship studies have been performed on these moieties. We designed and synthesized several analogs containing these structural motifs and evaluated their inhibition potencies against human FAAH enzyme. In addition, we built and validated a homology model of human FAAH enzyme and performed docking experiments. We identified several inhibitors in the low nanomolar range and calculated their ADME predicted values. These FAAH inhibitors represent promising drug candidates for future preclinical in vivo studies.     

Inhibition of human neutrophil chemotaxis by endogenous cannabinoids and phytocannabinoids: evidence for a site distinct from CB1 and CB2

Here, we show a novel pharmacology for inhibition of human neutrophil migration by endocannabinoids, phytocannabinoids, and related compounds. The endocannabinoids virodhamine and N-arachidonoyl dopamine are potent inhibitors of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced migration of human neutrophils, with IC(50) values of 0.2 and 8.80 nM, respectively. The endocannabinoid anandamide inhibits human neutrophil migration at nanomolar concentrations in a biphasic manner. The phytocannabinoid (-)-cannabidiol is a partial agonist, being approximately 40 fold more potent than (+)-cannabidiol; abnormal-cannabidiol is a full agonist. Furthermore, the abnormal-cannabidiol (CBD) analog trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-methyl-1,3-benzenediol (O-1602) inhibits migration, with an IC(50) value of 33 nM. This reported profile of agonist efficacy and potency parallels with the pharmacology of the novel "abnormal-cannabidiol" receptor or a related orphan G protein-coupled receptor, which are already known to modulate cell migration. Although having no effect alone, N-arachidonoyl l-serine attenuated inhibition of human neutrophil migration induced by anandamide, virodhamine, and abnormal-CBD. Our data also suggest that there is cross-talk/negative co-operativity between the cannabinoid CB(2) receptor and this novel target: CB(2) receptor antagonists significantly enhance the inhibition observed with anandamide and virodhamine. This study reveals that certain endogenous lipids, phytocannabinoids, and related ligands are potent inhibitors of human neutrophil migration, and it implicates a novel pharmacological target distinct from cannabinoid CB(1) and CB(2) receptors; this target is antagonized by the endogenous compound N-arachidonoyl l-serine. Furthermore, our findings have implications for the potential pharmacological manipulation of elements of the endocannabinoid system for the treatment of various inflammatory conditions.     

Synthesis and quantitative structure-activity relationship of fatty acid amide hydrolase inhibitors: modulation at the N-portion of biphenyl-3-yl alkylcarbamates

Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (FAAH IC50 = 63 nM) with a selected set of substituents of different size, shape, flexibility, and lipophilicity. Docking experiments and linear interaction energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the beta-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3'-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors.     

Cyclohexylcarbamic acid 3'- or 4'-substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors: synthesis, quantitative structure-activity relationships, and molecular modeling studies

Fatty acid amide hydrolase (FAAH) is a promising target for modulating endocannabinoid and fatty acid ethanolamide signaling, which may have important therapeutic potential. We recently described a new class of O-arylcarbamate inhibitors of FAAH, including the cyclohexylcarbamic acid biphenyl-3-yl ester URB524 (half-maximal inhibitory concentration, IC(50) = 63 nM), which have significant anxiolytic-like properties in rats. In the present study, by introducing a selected group of substituents at the meta and para positions of the distal phenyl ring of URB524, we have characterized structure-activity profiles for this series of compounds and shown that introduction of small polar groups in the meta position greatly improves inhibitory potency. Most potent in the series was the m-carbamoyl derivative URB597 (4i, IC(50) = 4.6 nM). Furthermore, quantitative structure-activity relationship (QSAR) analysis of an extended set of meta-substituted derivatives revealed a negative correlation between potency and lipophilicity and suggested that small-sized substituents may undertake polar interactions with the binding pocket of the enzyme. Docking studies and molecular dynamics simulations, using the crystal structure of FAAH, indicated that the O-biphenyl scaffold of the carbamate inhibitors can be accommodated within a lipophilic region of the substrate-binding site, where their folded shape mimics the initial 10-12 carbon atoms of the arachidonyl moiety of anandamide (a natural FAAH substrate) and methyl arachidonyl fluorophosphonate (a nonselective FAAH inhibitor). Moreover, substituents at the meta position of the distal phenyl ring can form hydrogen bonds with atoms located on the polar section of a narrow channel pointing toward the membrane-associated side of the enzyme. The structure-activity characterization reported here should help optimize the pharmacodynamic and pharmacokinetic properties of this class of compounds.     

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study

The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7+/-0.2 microm and Vmax=3.3+/-0.3 pmol min-1 (10(6) cells)-1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1-8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator.British Journal of Pharmacology (2004) 141, 457-467. doi:10.1038/sj.bjp.0705628     

Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.     

Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis--a difficult issue to handle

There is considerable debate at present as to whether the uptake of anandamide (AEA) into cells is by a facilitated transport process or by passive diffusion driven by fatty acid amide hydrolase (FAAH). The possibility that both processes occur, but to different extents depending upon the cell type used, has been difficult to investigate pharmacologically since available compounds show little selectivity between inhibition of AEA uptake and inhibition of FAAH. Recently, three compounds, UCM707 [N-(Fur-3-ylmethyl)arachidonamide], OMDM-1 and OMDM-2 [the 1'-(S)- and 1'-(R)-enantiomers of the 1'-4-hydroxybenzoyl analogue of oleoylethanolamide], selective for the uptake process, have been described and we have used these compounds, together with AM404 [(N-(4-hydroxyphenyl) arachidonoyl amide)] and VDM11 [(5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide]), with the initial aim of determining which mechanism of uptake predominates in C6 glioma and RBL-2H3 cells. AM404 and VDM11 were both found to decrease the uptake of 2 microM AEA into cells (IC50 values 6-11 microM), but they also inhibited rat brain FAAH (IC50 values 1-6 microM). However, when using a different FAAH assay protocol, VDM11 was a much less potent FAAH inhibitor (IC50>50 microM) regardless of the cell type and animal species used. In contrast, we confirmed that UCM707, OMDM-1 and OMDM-2 were weak inhibitors of FAAH (IC50 values >50 microM) under all conditions used. However, their potency as inhibitors of AEA cellular accumulation appears to be largely dependent on the cell type and assay conditions used. In particular, the potency of UCM707 (IC50 value > or =25 microM) was considerably lower than the submicromolar potency previously reported for U937 cells. It is concluded that the cause/effect relationship between AEA uptake and hydrolysis cannot be investigated uniquely by using supposedly selective inhibitors of each process.     

Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597)

In the present article, we review the pharmacological properties of KDS-4103 (URB597), a highly potent and selective inhibitor of the enzyme fatty-acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In vitro, KDS-4103 inhibits FAAH activity with median inhibitory concentrations (IC(50)) of 5 nM in rat brain membranes and 3 nM in human liver microsomes. In vivo, KDS-4103 inhibits rat brain FAAH activity after intraperitoneal (i.p.) administration with a median inhibitory dose (ID(50)) of 0.15 mg/kg. The compound does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice KDS-4103 elicits significant, anxiolytic-like, antidepressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB(1) receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1,500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were observed in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1,500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.     

SAR and LC/MS studies of β-lactamic inhibitors of human fatty acid amide hydrolase (hFAAH): evidence of a nonhydrolytic process

The endocannabinoid hydrolyzing enzyme FAAH uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of Ser-Asp-His) to cleave its endogenous substrates. Because inhibiting FAAH has a clear therapeutic potential, we previously developed β-lactam-type inhibitors of hFAAH. Here, we report the synthesis of five novel derivatives (5-9) of our lead compound 1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-azetidin-2-one (4, IC(50) = 5 nM) obtained via the systematic replacement of one to three carbonyls by methylene groups. The SAR results showed that the imide, but not the lactam, function is essential to the inhibition of hFAAH. We also performed LC/MS analysis following incubation of our inhibitors with hFAAH or mouse liver. We demonstrated that hFAAH interacts with these β-lactam-type inhibitors but, unexpectedly, does not open the β-lactam moiety. This mechanism seems to be unique to FAAH because the β-lactam function of the inhibitors is hydrolyzed when they are incubated in the presence of the serine hydrolases expressed in the mouse liver. Finally, we confirmed these results by showing that a highly selective FAAH inhibitor (PF-750) does not prevent this hydrolysis by liver homogenates.     

Analgesic actions of N-arachidonoyl-serotonin, a fatty acid amide hydrolase inhibitor with antagonistic activity at vanilloid TRPV1 receptors

BACKGROUND AND PURPOSE: N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia. EXPERIMENTAL APPROACH: AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured. KEY RESULTS: AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM251, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone. CONCLUSIONS AND IMPLICATIONS: Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.     

Design, synthesis and biological evaluation of novel arachidonic acid derivatives as highly potent and selective endocannabinoid transporter inhibitors.

In the present work, we have designed and synthesized a series of arachidonic acid derivatives of general structure I which have been characterized as highly potent and selective inhibitors of anandamide transporter (IC(50) = 24-0.8 microM, K(i) > 1000-5000 nM for CB(1) and CB(2) cannabinoid receptors and vanilloid VR(1) receptor). Among them, N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide deserves special attention as being the most potent endocannabinoid transporter inhibitor (IC(50) = 0.8 microM) described to date.     

Fatty acid amide hydrolase inhibitors from virtual screening of the endocannabinoid system

The endocannabinoid system consists of two cannabinoid receptors (CB1 and CB2), endogenous ligands (endocannabinoids), and the enzymes involved in the metabolism of the endocannabinoids, including fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL). In the present study, virtual screening of MGL inhibitors was performed by utilizing a comparative model of the human MGL enzyme. All hit molecules were tested for their potential MGL inhibitory activity, but no compounds were found capable of inhibiting MGL-like enzymatic activity in rat cerebellar membranes. However, these compounds were also tested for their potential FAAH inhibitory activity and five compounds (2-6) inhibiting FAAH were found with IC50 values between 4 and 44 microM. In addition, the hit molecules from the virtual screening of CB2 receptor ligands (reported previously in Salo et al. J. Med. Chem. 2005, 48, 7166) were also tested in our FAAH assay, and four active compounds (7-10) were found with IC50 values between 0.52 and 22 microM. Additionally, compound 7 inhibited MGL-like enzymatic activity with an IC50 value of 31 microM.     

Structure-activity relationship of a new series of reversible dual monoacylglycerol lipase/fatty acid amide hydrolase inhibitors

The two endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), play independent and nonredundant roles in the body. This makes the development of both selective and dual inhibitors of their inactivation an important priority. In this work we report a new series of inhibitors of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Among them, (±)-oxiran-2-ylmethyl 6-(1,1'-biphenyl-4-yl)hexanoate (8) and (2R)-(-)-oxiran-2-ylmethyl(4-benzylphenyl)acetate (30) stand out as potent inhibitors of human recombinant MAGL (IC(50) (8) = 4.1 μM; IC(50) (30) = 2.4 μM), rat brain monoacylglycerol hydrolysis (IC(50) (8) = 1.8 μM; IC(50) (30) = 0.68 μM), and rat brain FAAH (IC(50) (8) = 5.1 μM; IC(50) (30) = 0.29 μM). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the therapeutic potential of reversible MAGL inhibitors.     

Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications

Fatty acid amide hydrolase (FAAH) plays an important role in nerve function by regulating the action of endocannabinoids (e.g., anandamide) and hydrolyzing a sleep-inducing factor (oleamide). Several organophosphorus pesticides and related compounds are shown in this study to be more potent in vivo inhibitors of mouse brain FAAH than neuropathy target esterase (NTE), raising the question of the potential toxicological relevance of FAAH inhibition. These FAAH-selective compounds include tribufos and (R)-octylbenzodioxaphosphorin oxide with delayed neurotoxic effects in mice and hens plus several organophosphorus pesticides (e.g., fenthion) implicated as delayed neurotoxicants in humans. The search for a highly potent and selective inhibitor for FAAH relative to NTE for use as a toxicological probe culminated in the discovery that octylsulfonyl fluoride inhibits FAAH by 50% at 2 nM in vitro and 0.2 mg/kg in vivo and NTE is at least 100-fold less sensitive in each case. More generally, the studies revealed 12 selective in vitro inhibitors for FAAH (mostly octylsulfonyl and octylphosphonyl derivatives) and 9 for NTE (mostly benzodioxaphosphorin oxides and organophosphorus fluoridates). The overall in vivo findings with 16 compounds indicate the expected association of AChE inhibition with acute or cholinergic syndrome and >70% brain NTE inhibition with delayed neurotoxic action. Surprisingly, 75-99% brain FAAH inhibition does not lead to any overt neurotoxicity or change in behavior (other than potentiation of exogenous anandamide action). Thus, FAAH inhibition in mouse brain does not appear to be a primary target for organophosphorus pesticide-induced neurotoxic action (cholinergic or intermediate syndrome or delayed neurotoxicity).